Vitamin K, a fat-soluble vitamin discovered by Henry Dam in 1929, is now known to catalyze the posttranslational carboxylation of ten residues of glutamic acid in a prothrombin precursor protein in the endoplasmic reticulum of mammalian liver to form gamma-carboxyglutamic acid (Gla) residues. The enzyme system responsible for this vitamin K-dependent carboxylation is membrane-bound and can be solubilized with a number of detergents. The precise nature of the biochemical mechanism by which carbon dioxide is introduced into peptide-bound glutamate is still unknown. The specific aims of this research project are 1) to purify the membrane-bound vitamin K-dependent Glu-peptide carboxylase to homogeneity and to study its physical-chemical properties; 2) to determine the mechanism by which vitamin K catalyzes the carboxylation of Glu-peptides to form gamma-carboxyglutamate-containing peptides; 3) to determine the mode of action of the coumarin anticoagulant drug, particularly warfarin, in affecting the vitamin K-dependent carboxylase system; and 4) to reassemble the warfarin-vitamin K carboxylase in artificial membranes from purified components. In order to achieve these goals, modern technology designed to purify membrane-bound proteins and to detect, isolate, and determine the structures of vitamin K intermediates will be employed. These include high-pressure liquid chromatography, gas-liquid chromatography coupled to mass spectrometry, and electron-spin resonance spectrometry.